Supplementary Figure 2: Retrograde machinery is required for cell spreading and adhesion.

(a) Box-and-Whisker plots show RPE-1 cell spreading areas at different times after adhesion on fibronectin-coated coverslips (1 representative of 3 independent experiments. 20 min.: ctrl-siRNA n = 31 cells, Rab6-siRNA n = 29 cells; 2 h: ctrl-siRNA n = 12 cells, Rab6-siRNA n = 26 cells; 24 h: ctrl-siRNA n = 18 cells, Rab6-siRNA n = 22 cells). The ends of the Whiskers are set at 1.5× Interquartile Range above the third quartile, and 1.5× Interquartile Range below the first quartile). (b) Cell size in suspension measured by flow cytometry. Numbers of independent experiments: HeLa n = 4, RPE-1 n = 6. (c) Quantification by Western blotting of phosphorylation levels of focal adhesion kinase (pFAK) (n = 3 and 2 independent experiments for synt16 siRNA and Rab6 siRNA, respectively) in HeLa cells. (d) Percentage of RPE-1 cells labeled with the focal adhesion marker vinculin at different times after adhesion (n = 5 independent experiments). (e) Average number of RPE-1 cells adherent to fibronectin-coated coverslips per field of a ×10 objective, after 1 h of adhesion and 3 washes with PBS⁺⁺ (1 representative of 2 independent experiments. ctrl-siRNA n = 13 fields, synt16-siRNA n = 19 fields, Rab6-siRNA n = 17 fields). (f) Number of RPE-1 cells after a 5 min incubation with trypsin (1 representative of 2 independent experiments. ctrl-siRNA n = 4 fields, synt16-siRNA n = 3 fields, Rab6-siRNA n = 5 fields). Means ± s.d., ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, unpaired t-test.